Method for removing manganese from water

ABSTRACT

A process for removal of manganese from water which includes the steps of (i) preparing a fluidized bed of particles (eg. magnetite) in a bioreactor capable of adsorbing a strongly adherent biofilm of microorganisms (e.g. pedomicrobium manganicum) metabolising manganese to provide an actively propagated biomass, and (ii) passing a stream of water through the fluidized bed where manganese is adsorbed by said biomass and is thus removed from the stream of water to provide a purified effluent of water exiting from the bioreactor.

FIELD OF THE INVENTION

THIS INVENTION relates to a method and apparatus for removal ofmanganese from water and in particular potable or drinking water.

BACKGROUND OF THE INVENTION

The presence of manganese in drinking water constitutes a problem formany water authorities both in Australia (references 26 and 48 referredto hereinafter in the LIST OF REFERENCES) and overseas (4,62) as a causeof manganese-related "dirty-water" in urban distribution systems.Manganese entering the distribution system accumulates as a blackmanganese oxide biofilm on pipe surfaces and causes consumer complaintswhen it sloughs off (4, 26, 48, 51, 52, 53, 62). In a chlorinateddrinking water system the manganese oxide may be deposited chemically ormay be accumulated by viable bacterial biofilms which develop in areaswith insufficient chlorination (48, 51, 52, 53).

The manganese-related "dirty water" is not associated with any knownhealth risk but the water is aesthetically unacceptable and causeseconomic losses by irreversible staining of washing, equipment,manufactured goods and swimming pools.

The problem is widespread in Australia with many cities and towns alongthe east coast from Cairns in North Queensland to Wyong and Woolongongin New South Wales experiencing problems. Many of these coastal townsrely on tourism as their major industry and are expected to maintainhigh standards for their tourist image. In 1985, the most affectedconsumer complaints reached as high as 870 per week (48, 51, 53).

Most water authorities aim through various water treatment strategies toreduce manganese in drinking water to the WHO and NHMRC recommendedlevel of 0.05 mg/l (41,62). The American Water Works Association goallevel is 0.01 mg/l (4).

A recent extensive study (48, 51, 52, 53) of the Gold Coast waterdistribution system has shown that manganese-related consumer complaintsoccur when manganese levels reach 0.02 mg/l and approaches 80 per weekwhen levels rise to 0.05 mg/l. These consumer complaints are only anindication of the total number of consumers affected.

Current water treatment methods for the removal of manganese and ironfrom raw water supplies are destratification and oxygenation of the rawwater supply (46,61) and chemical oxidation at the treatment plantfollowed by filtration (61). The most commonly used oxidants are KMnO₄,chlorine, chlorine dioxide and ozone (61).

A survey of treatment plants by Green (24) indicates that the use ofsand filters as a manganese removal reactor effectively restricts thefilter loading rate to about 5 mh⁻¹. Modern rapid sand filters aredesigned to operate at up to 9 mh⁻¹ (32). It is evident, therefore, thatif the economic benefits of high rate filtration are to be achieved forhigh manganese sources, then significant manganese removal must beachieved at treatment stages preceding filtration (32).

Manganese (II) is not removed by conventional water treatment processessuch as alum flocculation unless an oxidation step is included. The mostcommon oxidant is KMnO⁴ which converts Mn (II) to Mn (IV) and thiscolloidal precipitate is subsequently removed by filtration. There arepractical difficulties with this method as the rate and extent ofoxidation is dependent on factors such as the speciation of manganese,the characteristics of organics present and filter efficiency. Thesefactors are often beyond the control of the plant operator. On occasionsvery little manganese is removed at worst the concentration may behigher after treatment that in the raw water.

Recently, chlorine and chlorine dioxide have been used in the dual rolesof disinfection and oxidation (61).

Biological oxidation of manganese offers an alternative to chemicalmethods and is already being used to some extent in water treatment, butnot to its full potential.

At neutral pH, manganese, unlike iron, is not oxidized by oxygen alone.The oxidation of manganese in natural and destratified oxygenated waterstorages is due to part of the action of manganese-oxidisingmicroorganisms (22, 57, 55).

There exists in nature a variety of microorganisms such as bacteria andfungi which are capable of oxidising manganese (22, 25). Such organismsare ubiquitous in their distribution occurring widely in natural soiland water habitats. Some of these organisms are well adapted to anattached mode of growth.

Biological oxidation and removal of manganese has been shown to occur inrapid sand filters colonised by manganese-oxidising bacteria(6,14,15,38). In a comprehensive study (13) of sand filters om 21treatment plants in Germany it was shown that the bacteria involved inmanganese removal belong to the genera Hyphomicrobium, Leptothrix,Metallogenium, Siderocapsa and Siderocystis. These organisms appear tohave a superior ability to adhere to surfaces and to withstand the shearforces associated with flowing water. These organisms are frequently tobe found in association with iron-oxidising organisms such asGallionella, Leptothrix and together they contribute to rapid removal ofmanganese and iron.

Such biologically active sand filters can be operated at loading ratesof up to 13 mh⁻¹ (13) and 24 mh⁻¹ (38) and are therefore compatible withmodern water treatment requirements. Disadvantages associated with usingsand filters as packed-bed bioreactors include clogging and binding ofparticles as biomass develops (2). This results in reduced flow ratesand a reduction in biofilm surface area available for contact withmanganese. It has been shown that the oxidation of manganese occurs onextracellular polymeric slime on the surface of manganese-oxidisingbacteria (13,23). Binding of particles causes channelling to occur inthe packed-bed so that water passes through with inadequate treatment.These problems necessitate frequent periodic cleaning of filters byvigorous backwashing, which may result in the removal of active biofilmand time is necessary for the filter to re-establish its manganeseremoval efficiency (18). The utilisation of microorganisms in wastewater and sewage treatment is well established. Their utilisation in thetreatment of drinking water has not been widely exploited. Where theyare used in the removal of manganese and iron (6,13,15,38), the processis poorly understood and has not been developed to the same level oftechnology as for waste water treatment. Little is known about theenvironmental conditions which control the growth and metabolic rate ofmanganese-oxidising bacteria. There is a body of research in theliterature on the biochemical mechanism proposed for manganeseoxidation. The results are frequently conflicting and very dependent onthe organism studied (eg. 15,22).

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a method andapparatus for removal of manganese from water which alleviates theproblems of the prior art discussed above.

The process of the invention includes the steps of:

(i) preparing a fluidised bed of particles in a bioreactor capable ofadsorbing a strongly adherent biofilm of microorganisms capable ofmetabolising manganese to provide an actively propagated biomass; and

(ii) passing a stream of water through the fluidised bed wherein Mn²⁺ orcolloidal manganese from the stream of water is adsorbed by said biomassand is thus removed from the stream of water to provide a purifiedeffluent of water exiting from the bioreactor.

In accordance with the invention the development of a fluidised-bedbioreactor offers several advantages over conventional sand filters forthe removal of manganese. Such reactors rely on the growth ofimmobilised cultures on small particles suspended in a water column byup-flowing water (2). This technology offers high surface area tobiomass volume ratios and thus higher efficiency for a given volume ofreactor. An added advantage is that fluidised-beds expand to accommodategrowing biomass and to tend to be self cleaning and will not clog (2).

Small, dense, monosized support particles have been recommended foraerobic water treatment (2). Small particles are desirable because theygive a higher surface area of biomass and there will be less transferresistance. Unlike a packed-bed the particle size and density, and theflow velocity in a fluidised-bed are not independent variables. Inaccordance with the invention it is considered that the combination of adense particle and a strongly adherent biofilm of manganese-oxidisingbacteria could be exploited in a biotechnological process for theoxidation of manganese in water treatment. By using a continuousrecirculating fluidised-bed bioreactor it was considered possible toselect a particle size and flow velocity with a resultant shear forcewhich would ensure the dominance of the manganese-oxidising biofilm. Asuitable particle size for the particles is of the range 50 μm to 1000μm (ie 1 mm).

Several strains of Pedomicrobium manganicum have been isolated from awater distribution system biofilm (48, 49,51,52,53), and it has beenshown that this organism was able to withstand the shear forcesassociated with high flow rates (52).

Tyler and Marshall (58,59) previously showed that a similarmanganese-oxidising budding bacterium Hyphomicrobium sp. was thedominant organism in biofilm which developed in hydroelectric pipelinesin Tasmania, illustrating the suitablility of this type of organism fora fixed biofilm reactor. Hyphomicrobium differs from Pedomicrobium byits ability to use Cl compounds as a source of carbon and energy and itsability to utilise inorganic nitrogen sources. Pedomicrobium requiredcomplex organic carbon and nitrogen sources such as humic acids whichare commonly found in raw water sources (20,48). Tyler and Marshall'sculture (strain T37) of Hyphomicrobium has since lost the ability tooxidize manganese through repeated laboratory subculture and was notavailable for study. However, the fact that Hyphomicrobium strains aswell as the microorganisms referred to above in (13) may also be used tometabolise manganese show that the invention is not limited to strainsof P. manganicum and that the invention is applicable to anymicroorganisms which are capable of metabolizing manganese.

It is also believed that magnetite particles used in the Sirofloc waterpurification process (31) have the necessary density and surfacecharacteristics for a suitable support particle. In an Australian patent534238 (reference 3) it has been shown that microorganisms attachstrongly to magnetite without diminishing their capacity to functionmicrobiologically. Mac Rae and Evans (33,34) showed that magnetiterapidly adsorbed 95-99% of a variety of microbial cells from aqueoussuspensions.

The process of the invention includes the installation of a continuousrecycle fluidised-bed bioreactor (CRFB) for the oxidation of manganese(II) as the raw water enters the treatment plant. Such a process wouldoperate without the addition of expensive chemical oxidants. In awide-ranging study of Australian water resources to the end of thecentury, Garman (19) concluded that "the presence of iron and manganesein Australian waters is seen as a major expense for water treatmentcosts".

An additional advantage hypothesised was that the manganese (IV) formedwould be firmly bound to organic expolymeric substances either free oron the surface of cells. This material would be more easily removed byalum flocculation and/or filtration than chemically formed manganeseoxide alone.

The treated water would undergo normal disinfection by chlorination. Itshould be emphasised that the organisms are involved are harmlessaquatic organisms and pose no health threat (17).

It is considered that the process of the invention has significantlycontributed towards a better fundamental understanding of biologicalmanganese oxidation and will very likely result in a major improvementin the capacity of water treatment plants to reduce manganese toacceptable levels. The simplicity of the process studied also takes itsuitable for primary treatment of water in small communities.

As will be apparent from the description hereinafter methods weredeveloped for the immobilisation of Pedomicrobium manganicum cells onmagnetite particles and to use the immobilised cells in a continuousrecycle fluidised bioreactor (CRFB) for the removal of manganese fromwater. A model CRFB was operated for 22 weeks with removal rates ofgreater than 90% and up to 100% for Mn²⁺ concentrations in the range0.25 to 8.5 mg/l when operated at a residence time of 21 hours. Themajority of the manganese in the effluent was residual Mn²⁺ with onlylow levels of oxidised and adsorbed manganese. As was hypothesised thebulk of the oxidised manganese remained attached to the immobilisedcells in the fluidised column. The bioreactor approached maximum removalefficiency within a week compared with up to 15 weeks for sand filtersrelying on colonisation by natural populations of manganese oxidisingbacteria. The CRFB required minimal maintenance, did not clog or bindand therefore did not require backwashing which is a disadvantage withsand filters. The pH conditions were critical for manganese adsorption,oxidation and removal. Optimal conditions were found to be around pH7.8.The research showed that surface components of P. manganicum weresignificant reservoirs of Mn²⁺. At pH8 approximately 45% of the Mn²⁺ wasadsorbed passively to the surface MnO₂ and 55% to the extracellularcomponents which were most likely acidic polysaccharides. Theextracellular components stabilised the adsorption of Mn²⁺ to the cellsat low pH. Research showed for the first time that the extracellularacid polysaccharides of P. manganicum are also able to bind preformedcolloidal MnO₂ a property which may be exploited in the CRFB for removalof fine particulate MnO₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing isotherms for adsorption of P. manganicumstrains to 1% magnetite (211-246 μm).

FIG. 2 is a graph showing isotherms for adsorption of P. manganicumstrain UQM3067 to 1% magnetite (211-246 μm) at varying pH.

FIG. 3 is a graph showing isotherms for adsorption of P. manganicumstrain UQM3067 to 1% magnetite (211-246 μm) using mixing by stirring andshaking.

FIG. 4 is a graph showing the effect of mixing time on the adsorption ofP. manganicum UQM3067 to 1% magnetite (211-246 μm).

FIG. 5 is a graph showing isotherms for the adsorption of P. manganicumUQM3067 to 1% magnetite of various sizes and sources.

FIG. 6 is a graph of the effect of pH on the desorption of Mn²⁺ fromvarious sources of P. manganicum.

FIG. 7 is a graph showing binding of colloidal MnO² by cells of P.manganicum at pH7 and pH4.

FIG. 8 is a transmission electronmicrograph of an ultra-thin section ofP. manganicum containing colloidal MnO².

FIG. 9 shows EDX elemental analysis of colloidal MnO² deposits bound topolysaccharides of P. manganicum.

FIG. 10A is a schematic of a single continuous recycle fluidizedbioreactor (CRFB) and associated flow conduits.

FIG. 10B is a schematic of a number of CRFBs in sequence and associatedflow conduits.

FIG. 11 is a graph showing immobilization of P. manganidum cells inmagnetite particles (CRFB).

FIG. 12 shows various fluorescent micrographs of immobilized cells of P.manganicum on magnetite particles.

FIG. 13 is a graph showing removal of 1 mg/l Mn₂₊ immediately afterimmobilization of P. manganicum cells by the CRFB.

FIG. 14 is a graph showing effect of decreasing Mn²⁺ concentrations onMn²⁺ removal by the CRFB immediately after immobilization of cells.

FIG. 15 is a graph showing effect of increasing Mn²⁺ concentrations onMn²° removal by the CRFB immediately after immobilization of cells.

FIG. 16 is a graph showing effect of Mn²⁺ concentration on the removalrate of Mn²⁺ by the CRFB.

FIG. 17 is a graph showing effect of pH control at pH 7 on Mn²⁺ removalshowing changes in input Mn²⁺, residual Mn²⁺, total residual Mn, pH andEh.

FIG. 18 is a graph showing effect of pH control at pH 7 on Mn²⁺conversion showing changes in pH, input Mn²⁺ and effluent levels oftotal Mn, soluble Mn, adsorbed Mn and MnOx.

FIG. 19 is a graph showing effect of pH control at pH 8 and pH 7.8 onMn²⁺ removal showing changes in input Mn²⁺, residual Mn²⁺, totalresidual Mn, pH and Eh.

FIG. 20 is a graph showing the effect of pH control at pH 8 and pH 7.8on Mn²⁺ conversion showing changes in pH, input Mn²⁺, and effluentlevels of total Mn, soluble Mn, adsorbed Mn and MnOx.

FIG. 21 is a graph showing the effect of age on the removal of 2 mg/lMn²⁺ by the CRFB operated at an influent pH of 7 and a residence time of21 h showing changes in input Mn²⁺, residual Mn²⁺ effluent pH.

FIG. 22 is a graph showing the effect of age on the removal of 2 mg/lMn²⁺ by the CRFB operated at an influent pH of 7 and a residence time of21 h showing changes in input Mn²⁺, residual Mn²⁺ effluent pH.

FIG. 23 is a graph showing the effect of age on the conversion of 2 mg/lMn²⁺ by the CRFB operated at an influent pH of 7 and a residence time of21 h showing changes in input Mn²⁺ and effluent levels of total Mn,soluble Mn, adsorbed Mn and MnOx.

FIG. 24 is a graph showing the effect of age on the conversion 2 mg/lMn²⁺ by the CRFB operated at an influent pH of 7 and a residence time of21 h showing changes in input Mn²⁺ and effluent levels of total Mn,soluble Mn, adsorbed Mn and MnOx.

DETAILED DESCRIPTION OF THE INVENTION Experimental Results SECTION 1 TheImmobolization of Pedomicrobium mangamicum Cells on Magnetite ParticlesINTRODUCTION

Work in this section was undertaken to investigate the ability of P.manganicum strains to absorb magnetite particles of various sources andsizes. These investigations were a prerequisite to the immobolizationstudies conducted in the bioreactor in Section 4.

MATERIALS AND METHODS

Bacterial culture. The strain of microorganism used was Pedomicrobiummanganicum UQM 3067 previously isolated from a water distribution systemwith manganese-depositing biofilm (51).

Magnetite. Crude ore obtained from Commercial Minerals was processed bysieving to the required size of 212-300 μm diameter. The magnetiteparticles were treated by eight alternating magnetic field cycles (8AMF)using the method of Mac Rae and Evans (33,34). This treatment wascarried out by mixing 1 volume of untreated magnetite with 4 volumes of0.1M NaOH for 10 minutes followed by 4 ten minute washings withdistilled water using decantation and then final readjustment to pH4with 1M H₂ SO4. Decantation was facilitated by the use of a magnet tohold the magnetite in the base of the beaker. The magnetite was thenpassed through a demagnetising field (Eclipse AD960, England) todisperse the particles. This treatment process was repeated 8 times.

Immobilization jar tests. The adsorption of P. manganicum cells to 8AMFmagnetite particles was studied in jar tests prior to immobilization inthe CRFB. A 1% volume of settle magnetite was added to 200 ml cellsuspension in immobilization suspending medium (33) in a 250 ml beakerand stirred at 250 rpm with a paddle stirrer for 10 minutes to keep themagnetite suspended and mixed. After cell adsorption the magnetite wasallowed to settle for 2 minutes with the aid of a magnet. Dilutions ofcell suspensions were made before and after adsorption and triplicate0.1 ml samples were plated on PSM agar (20) to determine the number ofunadsorbed cells. Agar plates were incubated at 28° C. for 10 days.

RESULTS Adsorption of P. manganicum to Magnetite Particles

Magnetite. The magnetite preparations used in the study are listed inTable 1. Unexpectedly, it was not possible to obtain magnetite of therequired size commercially. Consequently considerable time and effortwas involved in processing crude ore in our own laboratories and sievingto the required size of approximately 200-300 μm diameter. Smaller sizesof 104-147, 147-211, and 211-246 μm were also studied in preparation forlater studies on the effect of particle size on manganese removal. Theisoelectric points of the preparations were in the range of 5.14 to5.82.

Selection of P. manganicum culture. Two cultures were selected on thebasis on manganese oxidation from the eight cultures of P. manganicumpreviously isolated (48,49,51). The strains selected were UQM3066 andUQM3067. The adsorption of cells to magnetite was carried out in jartests using the method of Mac Rae and Evans (33,34). The adsorption wasassessed by viable counts on PSM agar (20) of cells remainingunadsorbed. FIG. 1 shows the isotherms for the adsorption of thecultures to magnetite particles of 211-246 μm. There was a significantstatistical difference beween the two strains and strain UQM3067 wasselected for further study because it appeared to offer slightly betterloadings of magnetite particles at higher concentrations of cells.

It can be seen from FIG. 1 that only approximately 50% of the cells wereadsorbed. This value was considerably lower than that 99% obtained forother organisms on 5 μm particles by Mac Rae and Evans (33). The mostlikely explanation was the reduced magnetite surface area and theslightly reduced purity of magnetite resulting from the larger size ofparticle which contains some impurities. The isoelectric points(Table 1) support this as values of 6.5-7.0 would have been expected forhigh purity magnetite. Several experiments were conducted to show thatother factors in the adsorption process were not the cause. Theseincluded the effect of pH, mixing method and time.

Effect of pH. The previous experiments were conducted at pH 7 which isthe desirable value for the bioreactor treating natural waters. However,adsorption of cells was also tried at pH 6 to see if this slightlyacidic pH might improve adsorption. FIG. 2 shows that there was nosignificant difference between adsorption at pH 6 and pH 7 andsubsequent experiments were conducted at pH 7.

Effect of mixing method. A possible cause of the reduced adsorption wasthe shearing forces caused by the more vigorous stirring required tosuspend particles of the size studied compared with smaller 5 μmparticles examined previously by Mac Rae and Evans (33,34). To test thispossibility adsorption was also studied by more gentle mixingaccomplished by gently inverting the mixture 60 times per minute. FIG. 3shows that there was no significant difference between the two methods.

Effect of mixing time. A further possible cause of reduced adsorptionwas the nature of the growth of P. manganicum. This organism does notgrow as single cells but as a network of hyphae and budding cells. Itwas possible the vigorous stirring was breaking up clumps of cellsproducing an effectively higher count during mixing and thereforeartificially reducing the adsorption efficiency. However, the results inFIG. 4 show that the number of adsorbed cells remained constant overtime, as did the number of unadsorbed cells, and that mechanicaldisruption was not a significant factor.

Effect of source and particle size. Adsorption of P. manganicum to themagnetite particles listed in Table 1 was carried out. The resultspresented in FIG. 5 show that there is no practical difference inadsorption to magnetite from different sources or to magnetite particlesin the size range 104-300 μm diameter. There were, however, somesignificant statistical differences which are being examined further butthese are unlikely to effect the operation of a bioreactor.

Effect of growth medium. The adsorption experiments reported wereconducted by suspending the cells in the standardised suspending mediumof Mac Rae and Evans (33,34). In order to test the effect of the PCgrowth medium to be used in the bioreactor, experiments were carried outto determine if cells adsorbed when transferred to PC medium. Theresults presented in Tables 2 and 3 show that there was no significantdifference in the number of cells adsorbed when particles weretransferred to suspending medium of PC medium. In both cases the losswas about 26%. It can also be seen that the cells remaining were firmlyadsorbed and there was no significant difference in the number of cellsremaining adsorbed at 10 minutes compared with 30 seconds (Tables 4 and5).

Effect of repeated adsorption. Further experiments were conducted todetermine if the less than expected adsorption was due to reducedsurface area as postulated. A suspension of P. manganicum cells wastreated with magnetite particles. The unadsorbed cells were removed andretreated with fresh magnetite. This process was repeated four times.The results presented in Table 6 show that a reducing proportion ofcells was adsorbed with each subsequent treatment. This indicates thatmore than one factor is causing this result. Surface area is probably afactor but also it seems that a proportion of cells have surfacecharacteristics which are less susceptible to adsorption to magnetite.However, this is a minor proportion and after four adsorptions only 6%of cells remained unadsorbed.

SECTION 2 The Adsorption and Desorption of Mn²⁺ by Surface Components ofPedomicrobium manganicum INTRODUCTION

This research was undertaken because of a need to determine the amountof Mn²⁺ adsorbed to particulate manganese and to the surface ofPedomicrobium manganicum cells during studies on manganese oxidationrates under different enviromental conditions in the bioreactor. Twomajor extracellular components adsorb significant levels of Mn²⁺. Theseare the extracellular acidic polysaccharides which have been shown byGhiorse and Hirsch (23) to be intimately associated with manganeseoxidation in Pedomicrobium sp., and the manganese oxide deposited on thepolysaccharides (37).

It is thought that adsorption of Mn²⁺ to the extracellularpolysaccharides is a prerequisite to manganese oxidation by an as yetunidentified protein or enzyme (23). The adsorptive properties of MnO₂for Mn²⁺ and other divalent metal ions are well documented (37) and arerelated to its surface properties (28,36,39). The adsorption of Mn²⁺ toMnO₂ is pH independent (37) and because of the polyanionic nature ofacidic polysaccharides (27) it would be expected that the adsorption ofMn²⁺ to the polysaccharides would also be pH dependent. What was notknown is the contribution of each component or other components such assurface proteins to Mn²⁺ adsorption by P. manganicum and the desorptivebehaviour of each under changing pH conditions.

Desorption by ionic exchange using the divalent metal ions Mg²⁺ and Cu²⁺has been used to estimate the level of Mn²⁺ adsorbed to particulate MnO₂(1,7,8,11,30) bit the effectiveness of these methods for quantitativestudies with P. manganicum is not known.

This section reports the results of a series of experiments whichinvestigated the adsorption of Mn²⁺ by surface components ofPedomicrobium manganicum and the effect of pH and cationic exchange onMn²⁺ desorption.

MATERIALS AND METHODS

Microorganism. The strain used in these studies was Pedomicrobiummanganicum UQM3067 which was isolated from a drinking-water distributionsystem experiencing manganese-related dirty water problems (51).

Growth of P. manganicum. Strain UQM3067 was grown at 28° C. in 21 flaskscontaining 11 PC medium (58). PC medium contained 0.005% Bacto yeastextract (Difco) and 0.002% MnSO₄.4H₂ O in deionised water. Cells forMn²⁺ adsorption experiments were grown for 3 weeks until no residualMn²⁺ remained in the growth medium. The cells were harvested bycentrifugation at 7000 g for 10 minutes and washed twice with deioinsedwater before use.

Glassware. All glassware used was acid washed in 8N nitric acid and thenrinsed with high purity deionised water (milli-Q, Millipore Corporation,France).

Mn²⁺ adsorption experiments. Adsorption of Mn²⁺ (manganese nitratespectroscopy standard, BDH Chemicals) to MnO₂ -encrusted P. manganicumcells or abiological MnO₂ (precipitated β-MnO₂ (pyrolusite) AjaxChemicals) was carried out in 100 ml glass beakers. The surface area ofboth systems was approximately 0.25 m². P. manganicum were evenly coatedwith MnO₂ and it has been assumed that the surface area is a measure ofexposed MnO₂ surface on the cells. Washed cell pellets (0.25 g wet wt.)containing 464 μg MnO₂ were resuspended in 0.5 ml deioised water andadded to 50 ml maganese nitrate solution containing approximately 80 μgMn²⁺ and previously adjusted to pH 8 with soium hydroxide. The mixturewas stirred gently with a magnetic stirrer at room temperature for 5minutes after which Mn²⁺ adsorption was complete. In experiments usingabiological MnO₂ the microbial cells were substituted by an equivalentamount of finely ground abiological MnO₂ with the same surface area.Samples (5 ml) of adsorption mixtures were taken and filteredimmediately through 0.1 μm membrane filters (Sartorius GmbH, W.Germany). The filtrates were analysed to determine unadsorbed Mn²⁺.

Mn²⁺ adsorption inhibition experiments. Various treatments of P.manganicum cells designed to inhibit metabolic activity or adsorption toextracellular proteins or polysaccharides were undertaken to determinetheir effect of Mn²⁺ adsorption. The treatments included: autoclaving(121° C./15 min.), steaming (100° C./10 min.) (9), EDTA extraction (9),Zwittergent extraction (14), 2M NaOH extraction (9), 10 mM NaN₃ (30), 1mM HgCl₂ (23), 0.05% (w/v) glutaraldehyde (23), and protease (SigmaP-5147, 0.3 units/ml). Treated cells were studied as described above fornormal viable cells with the addition of inhibitors to the mixtureswhere appropriate.

Mn²⁺ desorption experiments. After adsorption of Mn²⁺ at pH 8 the effectof pH on Mn²⁺ desorption was followed by adjusting the pH of thereaction mixtures in stepwise increments from 8 down to 2 by theaddition of nitric acid. Samples (5 ml) were taken at each pH intervaland filtered immediately through 0.1 μm membrane filters (SartoriusGmbH, W. Germany). The filtrates were analysed to determine unadsorbedMn²⁺. Control experiments were included to correct for any Mn²⁺desorption from P. manganicum cells or abiological Mn²⁺.

Determination of Mn²⁺ adsorbed to P. manganicum. Various publishedtreatments were used to compare their effectiveness for Mn²⁺ desorptionfrom MnO₂ -encrusted cells of P. manganicum. The reagents used were 10mM MgSO₄, pH 4.2 (8) and 10 mM CuSO₄ in 1M ammonium acetate, pH 7 (8).Each treatment was tested on samples at pH 2, 4 and 7. A 5 ml volume ofcell pellet to which approximately 7 μg of Mn²⁺ had been adsorbed wasmixed with 5 ml of desorption reagent. The mixtures were gently agitatedfor 4 h and then filtered through a 0.1 μm membrane filter (SartoriusGmbH, W. Germany). The filtrates were analysed to determine the Mn²⁺desorbed. The difference between CuSO₄ - or MgSO₄ -treated cells andcontrol water-treated cells was used to determine the amount of Mn²⁺desorbed and by calculation the amount of Mn²⁺ adsorbed to the cellpellet (30). The concentration of soluble Mn in the filtrates wasdetermined by flame atomic absorption spectrophotometry.

Analysis of Mn. The porphyrin colorimetric method of Ishii et al. (29)was used to determine Mn²⁺ concentrations in filtrates by measuringabsorbance at 469 nm in 4 cm light-path curvetres in a Unicam SP600spectrophotometer (PYE Unicam Ltd., U.K.) and comparing with a standardcurve using dilutions of manganese nitrate spectroscopy standard (BDHChemicals). In the CuSO₄ desorption experiments manganese was determinedby flame atomic absorption spectrophotometry using a Varian AA875spectrometer because of interference with the prophyrin colorimetricmethod by the presence of CuSO₄. Flame atomic absorptionspectrophotometry was also used to determine MnO₂ concentrations in cellpreparations after digestion in 11M HCl.

Determination of surface area. Surface area analyses were carried out bythe gas adsorption technique (17) with a Micromeritics R5AA 2205 surfacearea analyser using argon as the asorbate.

RESULTS

Adsorption of Mn²⁺ to P. manganicum. The results of experimentspresented in Table 7 show that viable cells of MnO₂ -encrusted P.manganicum adsorbed more than twice as much Mn²⁺ at pH 8 as cells killedby autoclaving at 121° C. for 15 minutes or steaming at 100° C. for 10minutes. The heat-killed cells adsorbed approximately the same amount ofMn²⁺ as abiological MnO₂ with the same surface area. The treatment ofviable P. manganicum cells with 10 mM NaN₃ to inhibit metabolic activitydid not reduce the capacity of the cells to adsorb Mn²⁺. Similarly,treatment of cells with 1 mM HgCl₂, 0.05% glutaraldehyde or 0.3 units/mlprotease to denature extracellular enzymes or proteins had no effect ofMn²⁺ adsorption. Of the treatments chosen to denature extracellularpolysaccharides, steaming at 100° C. for 10 minutes had a marked effect,and 2M NaOH had a slight effect. The experiments also showed that oxygenwas not required for the adsorption of Mn²⁺ to P. manganicum cells.

Effect of pH on Mn²⁺ desorption from P. manganicum. In these experimentsa comparison was made between the desorption of Mn²⁺ from viable andkilled (autoclaved) cells of MnO₂ -encrusted P. manganicum, andabiological cell-free MnO₂, each system having the same surface area. Ascan be seen from the data in Table 7 and FIG. 6, the autoclaved cells ofP. manganicum had a similar adsorptive capacity for Mn²⁺ as abiologicalMnO₂ whereas viable cells and sodium azide-treated cells had more thantwice the adsorptive capacity.

The desorption experiments presented in FIG. 6 showed two distinct pHdesorption profiles. The experiments showed for abiological MnO₂ that asthe pH was reduced no Mn²⁺ was desorbed between pH 8 and pH 6 afterwhich there was a steady desorption until this was complete byapproximately pH 4 although the desorption occurred linearly over theentire pH 8 to pH 4 range. No dissolution of MnO₂ occurred at pH 2 overthe period of the experiments.

In contrast to the desorption profile for abiological MnO₂, viable cellsof P. manganicum exhibited an exponential desorption profile below pH 6and sodium azide-treated cells behaved in a similar manner. Autoclavedcells behaved essentially as abiological MnO₂. However, whereasdesorption from autoclaved preparations was complete at pH 4, viablecells were able to bind considerable amounts of Mn²⁺ below pH 4 (Table8) and desorption was not complete even at pH 2 (Table 9).

Determination of Mn²⁺ adsorbed to P. manganicum. The results presentedin Table 9 confirm that the CuSO₄ (pH 4.2) method of Bromfield and David(7) for the desorption of adsorbed Mn²⁺ on particulate MnO₂ is the mosteffective. A maximum of 95.7% of the Mn²⁺ adsorbed to the P. manganicumcells was recovered and the desorption efficiency was shown to the veryreproducible with recoveries of 95.7 ±0.78% over eight replicateanalysis. The addition of ammonium acetate to the CuSO₄ reagent reducedthe desorption efficiency by 10%. MgSO₄ performed poorly as an aid toMn²⁺ desorption with only 2-3% of the Mn²⁺ desorbed at pH 4. In deioisedwater, lowering the pH to 2 alone resulted in the desorption of 90.5% ofthe adsorbed Mn²⁺. However, the addition of CuSO₄ at pH 2 reduced thedesorption efficiency.

SECTION 3 The Binding of Colloidal MnO₂ by Extracellular Polysaccharidesof Pedomicrobium manganicum INTRODUCTION

Microorganisms make a significant contribution to the natural cycling ofmanganese (22,42,43). A wide variety of microorganisms have been shownto oxidise or reduce the oxidation states of manganese(5,20,22,25,42,51,58). Ghiorse and Hirsch (23) demonstrated that theoxidation and deposition of manganese by the budding hyphal bacteriumPedomicrobium sp. occurred on extracellular polysaccharides. Thisobservation suggested a mechanism involving the adsorption of Mn²⁺ tothe extracellular polysaccharides followed by its oxidation to manganeseoxide catalysed by an as yet unidentified agent. However, ourobservation of manganese oxide deposition by Pedomicrobium manganicumsuggested that this organism may also be able to bind and depositpreformed manganese oxide in the absence of Mn²⁺.

This section reports the results of a series of experiments designed totest the ability of the extracellular polysaccharides of P. manganicumto bind and deposit colloidal MnO₂.

METHODS AND MATERIALS

Microorganisms. The strain used in these studies was Pedomicrobiummanganicum UQM 3067 which was isolated from a drinking-waterdistribution system experiencing manganese-related dirty water problems(51).

Growth of P. manganicum. Strain UQM 3067 was grown at 28° C. inPedomicrobium standard medium (PSM) (20) containing 10 mM sodiumacetate, 0.5 g/l Bacto yeast extract (Difco), vitamin supplement andmineral salts base adjusted to pH9. The mineral salts base 912)contained per liter: ethylene diamine tetra-acetic acid (2.5 mg),Zn2O₄.7H₂ O (11 mg), FeSO₄ 7 H₂ O (5 mg) MnSO₄ H₂ O (1.54 mg), CuSO₄ 5H₂O (0.39 mg), Co(NO₃)₂ 6H₂ O (0.25 mg), and Na₂ B₄ O₇ 10H₂ O (0.18 mg).For binding experiments, cells were grown for 7 days in PSM broth,harvested by centrifugation at 7,000 g for 15 min. and washed twice withdeionised water before use.

Glassware. All glassware used was acid washed in 8N nitric acid and thenrinsed with high purity deionised water (Milli-Q, Millipore Corporation,France).

Preparation of colloidal MnO₂. Colloidal MnO₂ suspensions were preparedat room temperature by slowly adding a slight stoichiometric excess ofpotassium permanganate solution (8 ml, 40 mg Mn/L) to 150 ml manganoussulphate (1.33 mg Mn/L) while stirring. The mixture was made up to 200ml with high purity deionised water and aged for 1 month until all theMn²⁺ was oxidised and the excess permanganate decomposed. The oxidationstate of the colloidal MnO₂ was confirmed by iodometric titration (40)which gave a value of 4.08. Prior to use the colloidal MnO₂ was filteredthrough a 0.45 μm membrane filter and the concentration determined byflame atomic absorption spectrophotometry (AAS).

Binding of colloidal MnO₂. Washed cell pellets (0.2 g wet wt.) of P.manganicum were suspended in 5 ml deionised water and added to 400 mlcolloidal MnO₂ suspension containing approximately 1 mg Mn/L andpreviously adjusted to the experimental pH with 0.1N NaOH for pH 7experiments and 0.1N HNO₃ for pH 4, 5 and 6 experiments. The pH wasmonitored during the course of each binding experiment. No pH changeoccurred at pH 6 and 7, and there was a 0.5 pH unit increase at pH 4 and5 after 24 h. The mixture was stirred gently with a magnetic stirrer.The rate of binding was followed by monitoring the concentration ofcolloidal MnO₂ remaining in suspension. Control experiments were carriedout without bacterial cells to take account of any aggregation ofcolloidal particles. Ten ml samples of the mixture were taken at 30 min.intervals for 5-7 hours and at 24 hours and filtered through 0.45 μmmembrane filters. The concentration of manganese in the filtrates wasdetermined by flame AAS using a Varian AA 875 atomic absorptionspectrometer fitted with a graphite furnace. The apparatus wascalibrated by use of standard manganese solutions obtained by dilutionof a 1000 ppm spectroscopy standard solution (BDH).

Electron microscopy. After the completion of the binding experiments thecells of P. manganicum were recovered from the mixture by centrifugationat 2000 g for 10 min. and washed with deionised water to remove anyunbound MnO₂. The cells were then fixed with 3% glutaraldehyde in 0.1Mcacodylate buffer (pH 7.4) at room temperature. After 2 h the cells werewashed 3 times in 0.1M cacodylate buffer and stored at 4° C. overnight.The cells were recovered by centrifugation and post-fixed at pH 7.4 for2 h at 4° C. in 1% OsO₄ to which 5% ruthenium red (Johnson Matthey,London) was added to a final concentration of 0.05% (v/v). The cellswere then immobilised in 2% agarose and the agar blocks dehydrated inethanol and embedded in LR white medium grade resin (Bio-Rad, USA) (44).Polymerization of the resin was carried out for sequential 2 h, 4 h and4 h periods at 50° C. under a nitrogen atmosphere and then overnight at4° C. Thin sections were cut with a diamond knife using a Sorvall MT5000 ultramicrotome and picked up on nitrocellulose coated copper grids.Thin sections on grids were stained with 4% uranyl acetate followed by1.2% lead citrate (57) before examination in an Hitachi transmissionelectron microscope (Model H-800).

Energy dispersive X-ray microanalysis (EDAX). Semi-thin sections 700-800nm thick on copper grids were stained as before and examined by EDX forthe presence and location of manganese deposits using a JEOLtransmission electron microscope (Model JSM-35 CF) fitted with a TracerNorthern X-ray analyser (Model TN 4000).

RESULTS

Binding of colloidal MnO₂. The results of experiments presented in FIG.7 show that cells (0.2 g, wet wt.) of P. manganicum were able to bindcolloidal MnO₂. There was an initial rapid binding followed by a slowerlinear binding rate which extended over several hours. The initialbinding level, the linear binding rate, and the total binding capacitywere pH dependent (Table 10, FIG. 7). Only approximately 10% of thecolloidal MnO₂ was bound at pH7 and pH6, but as the pH was loweredfurther, the binding capacity increased with a sharp rise between pH5and pH4. At pH4, the cells bound 54% of the MnO₂ after 2 min. comparedwith 10.9% at pH7. After 150 min. at pH4, the level of MnO₂ bound hadrisen to 88% but this fell away to 54.8% after 24 h. This remaining MnO₂was stably bound and no further desorption or detachment occurred onstanding for a further 24 h or longer. The MnO₂ bound at pH4 was pHstable and remained bound to the cells even after increasing the pH to5, 6 or 7 for 24 h.

Mechanism of MnO₂ binding. Transmission electron-microscopy ofultra-thin sections of P. manganicum cells showed that the colloidalMnO₂ was bound to extracellular polymers (FIG. 8). The positive stainingof these polymers by ruthenium red indicated that they were acidicpolysaccharides. Ribbon-like particulate deposits typical of theappearance of δ-MnO₂ were bound to the surface of the extracellularacidic polysaccharides (FIG. 8). Elemental analysis of the bounddeposits by EDAX confirmed the presence of manganese (FIG. 9). The heavymetals )s, Pb and U whose salts were used for staining the ultra-thinsections were observed to be strongly adsorbed to the MnO₂ deposits. Theorigin of the Cr is uncertain but was possibly a trace contaminant ofthe heavy metal salts. Electron microscopy (not shown) also confirmedthat substantially more colloidal MnO₂ was bound to cells at ph4 than atpH7.

SECTION 4 The Adsorption and Oxidation of Manganese by Immobilised Cellsof Pedomicrobium manganicum INTRODUCTION

Work presented in the previous sections demonstrated that it waspossible to immobilise cells of P. manganicum on magnetite particles andthe P. manganicum cells were capable of adsorbing and binding Mn²⁺ andMnO₂ respectively to surface components. Research in this sectiondemonstrates that these properties can be exploited in a fluidised bedbioreactor to oxidise and remove Mn²⁺ from water.

Disadvantages associated with using sand filters as packed-bedbioreactors include clogging and binding of particles as biomassdevelops (2).

In this section we report the results of research on a modelfluidised-bed bioreactor for the adsorption and oxidation of manganese.The model utilises cells of the manganese-oxidising bacteriumPedomicrobium manganicum immobilised on particles of magnetite. P.manganicum has ideal characteristics for this application. Previousresearch has shown that P. Manganicum adheres strongly to surfaces andwithstands high shear forces in water distribution systems (52), andactively adsorbs and oxidises manganese on extracellular components. Inaddition the extracellular acidic polysaccharides bind colloidal MnO₂(50). Magnetite particles have been shown to rapidly adsorb a variety ofmicroorganisms (33,34) without diminishing their metabolic activity (3),thus potentially reducing the start up period required when said is usedas a support medium.

MATERIALS AND METHODS

Continuous recycle fluidized bioreactor (CRFB). The model CRFB (FIG. 10)consisted of a glass column 60 mm in diameter and 600 mm high throughwhich medium was pumped into the bottom to fluidize 1.2 l magnetiteparticles of 212-300 μm diameter. The 50% expanded fluidized bed wasmaintained by recirculating a 3.3 l volume of medium through the columnand a stirred mixing vessel containing 2.1 l which was aerated by 1liter air per minute. Probes for pH, redox, and dissolved oxygen whereincluded in the mixing vessel and temperature was maintained at 25° C.Dual synchronised peristatic pumps were used to recirculate the mediumat a rate of 1 liter per min into and out of the column and mixingvessel. When operating in continuous mode synchronised peristatic pumpswere used to pump in fresh medium and remove the same volume of effluentfrom the mixing vessel. The growth medium used was half strength PCmedium (58) containing 0.0025% yeast extract. The total organic carboncontent was determined as 12 mg/l. Manganese concentration was variedfrom 0.25 to 8.5 mg/l.

With specific reference to FIGS. 10A and 10B, the CRFB 10 includes along column 11 having a bottom inlet 12 and top part 13 of enlargedcross-section compared to the column 11. There is also providedperistatic pumps 14 and 15, liquid flow gauge 16, air flow gauge 17,mixing vessel 18, agitator 19 and bubble catcher 20. Effluent may bedischarged from the top part 13 of the CRFB 10 as shown by line 21 inphantom or alternatively from mixing vessel 18 as also indicated by line22 shown in phantom. Influent may enter mixing vessel 18 suitablethrough line 23 and air may enter the mixing vessel through line 24through air flow gauge 17. Liquid may pass out of mixing vessel 18through line 25, bubble catcher 20 and pump 15. Liquid may also passfrom top part 13 of CRFB 10 and through pump 14 and air flow gauge 16,mixing vessel 18 through line 26.

In the arrangement shown in FIG. 10B, the mixing vessel 18A has influententering through line 23 and air also entering through line 24 throughair flow gauge 17. Also provided are peristatic pumps 14A and 15A withliquid entering the bottom inlet 12 of CRFB 10A and also exiting fromtop part 13 as an overflow through line 26 or also through line 27 tomixing vessel 18B and CRFB 10B wherein the process may be repeatedwhereby the overflow through line may proceed to further CRFBs (notshown).

Bacterial culture. The strain of microorganism used was Pedomicrobiummanganicum UQM 3067 previously isolated from a water distribution systemwith manganese-depositing biofilm (51).

Magnetite. Crude ore obtained from Commercial Minerals was processed bysieving to the required size of 212-300 μm diameter. The magnetiteparticles were treated by eight alternating magnetic filed cycles (8AMF)sing the method of Mac Rae and Evans (33, 34). This treatment wascarried out by mixing 1 volume of untreated magnetite with 4 volumes of0.1M NaOH for 10 min followed by four ten minute washings with distilledwater using decantation and then final readjustment to pH4 with 1M H₂SO₄. Decantation was facilitated by the use of a magnet to hold themagnetite in the base of the beaker. The magnetite was then passedthrough a demagnetising field (Eclipse AD960, England) to disperse theparticles. This treatment process was repeated 8 times.

Immobilization jar tests. The adsorption of P. manganicum cells to 8AMFmagnetite particles was studied in jar tests prior to immobilization inthe CRFB. A 1% volume of settled magnetite was added to 200 ml cellsuspension in immobilization suspending medium (33) in a 250 ml beakerand stirred at 250 rpm with a paddle stirrer for 10 min to keep themagnetite suspended and mixed. After cell adsorption the magnetite wasallowed to settle for 2 min with the aid of a magnet. Dilutions of cellsuspensions were made before and after adsorption and triplicate 0.1 mlsamples were plated on PSM agar (20) to determine the number ofunadsorbed cells. Agar plates were incubated at 28° C. for 10 days.

Immobilisation of cells in the CRFB. P. manganicum cells were harvestedby centrifugation from a 4.5 l culture grown for 2 weeks at 28° C. inPSM broth medium (20). The cell pellet was washed twice in sterileimmobilization suspending medium (33).

The CRFB was filled with 3.3 l sterile immobilization suspending medium.The washed cells were added to the mixing vessel which was stirred at500 rpm to break up any cell clumps. To immobilize the cells therecirculation pump was turned on to fluidize the magnetite and tocirculate the cells through the CRFB at a rate of 1 l per min.Immobilization was monitored by following absorbance at 540 nm and bytaking samples for viable cell counts. After 20 min. two changes ofsuspending medium were made to remove unadsorbed cells and then the CRFBwas filled with medium containing 1 mg/l Mn²⁺.

Analysis of manganese. The porphyrin colorimetric method of Ishii et al,(29) was used to determine Mn²⁺ concentrations in filtrates by measuringabsorbance at 469 nm in 4 cm light-path cuvettes in a Unicam SP600spectrophotometer (PYE Unicam Ltd, U.K.) and comparing with a standardcurve using dilutions of manganese nitrate spectroscopy standard (BDHChemicals), In CuSO₄ desorption experiments manganese was determined byflame atomic absorption spectrophotometry (AAS) using a Varian AA875spectrometer because of interference with the porphyrin colorimetricmethod by the presence of CuSO₄. AAS was also used to determine totalmanganese and MnO₂ concentrations after digestion of samples in 11M HCl.

Adsorbed Mn²⁺ was determined by,the method of Bromfield and David (7),Samples were adjusted to pH4 using 0.1N HNO₃ and mixed with an equalvolume of 10 mM CuSO₄ (pH 4.2) reagent to desorb adsorbed Mn²⁺. Themixture was allowed to stand for 4 h an then filtered through a 0.1 μmmembrane filter. Total Mn²⁺ in the filtrate was determined by AAS andadsorbed Mn²⁺ and residual Mn²⁺ determined as above without CuSO₄treatment.

Determination of surface area. Surface area analyses were carried out bythe gas adsorption technique (10) with a Micromeretics RSAA 2205 surfacearea analyser using argon as the asorbate.

Total organic carbon. Samples were filtered through 0.1 μm membranefilters and then analysed in an ASTRO TOC Analyser.

Viable counts. Viable counts of P. manganicum were made from multipledilutions (10⁻², 10⁻⁴, 10⁻⁶) of effluent samples and spread-platingtriplicate 0.1 ml aliquots of each dilution on PC agar (58) plates. Thegar plates were incubated at 28° C. for 14 days and examined formanganese-oxidizing bacteria. Viable counts were expressed as colonyforming units (cfu).

Electron microscopy. The adsorption and adhesion of cells of P.manganicum to the surface of magnetite particles was examined byscanning electron microscopy. Magnetite particles from jar tests or fromthe fluidized bed of the CRFB were fixed in 1% glutaraldehyde in 0.1Mphosphate buffer for 2 h and then dehydrated in a 25, 50, 85, 95 and100% ethanol series. The particles were transferred to a 50%amylacerate-ethanol mixture and then into amylacerate. The preparationswere critical print dried, sputter coated with gold in a partial argonatmosphere, and examined in a Philips SEM 505 scanning electronmicroscope.

Fluorescence microscopy. The attachment of cells to magnetite particleswas observed by fluorescent microscopy with an Olympus BHB fluorescentmicroscope after staining with acridine organ (5 -μg/ml).

RESULTS

Adsorption of P. manganicum to magnetite. Research presented in Section1 demonstrated that it wa possible to adsorb cells to magnetiteparticles and that the cells adhered strongly. The results presented inTable 11 demonstrate the adsorption of cells to volumes of magnetite ofthe ratio to be use din the CRFB. The results how that it was possibleto scale up the magnetite volume but that there was a reduction inparticle coverage from approximately 27 cells/10⁴ μm² at 1% magnetite to1 cell/10⁴ μm² at 45% magnetite. A jar test to mode the CRFB using 36%magnetite (Table 12) estimated that he fluidized bed of the bioreactorwould be loaded with 5.7×10⁶ cells epr ml magnetite with a coverage of 3cells/10⁴ μm².

Immobilisation of P. manganicum. Immobilisation of cells in the CRFBclosely followed the predictions of the jar tests. The jar test (Table12) predicted a loading of 6.8×10" cells on the 1200 ml of fluidizedmagnetite in the CRFB. The actual result was 1.6×10" which was of thesame order and provided a coverage of about 1 cell/10⁴ μm².Immobilization occurred rapidly and was complete in 5 min. (FIG. 11) aspredicted. Observations by fluorescent microscopy and scanning electronmicroscopy confirmed the immobilization of the cells on the magnetiteparticles (FIG. 12). The majority of cells were located in depressionsin the magnetite particulars indicating that the fluidizing conditionsof approximately 20 m/h may be too high for an even coverage because ofthe abrasive action of colliding particles.

Bioreactor startup. Immediately after immobilisation the CRFB wasflushed with immobilisation medium to remove unadsorbed cells and thenfilled with medium containing 1 mg/l Mn²⁺. The CRFB was operated inbatch mode for four days. The residual Mn²⁺ was reduced to zero in 3days with approximately 90% of residual Mn²⁺ being removed on eachsuccessive day (FIG. 13). The CRFB was challenged with a 5 h pulse of 1mg/l Mn²⁺ at a feed rate of 158 ml/h (=21 h residence time). Theresidual Mn²⁺ rose to 53 μg/l and then fell away to zero in 3 hours.

The CRFB was then operated continuously with a 1 mg/l Mn²⁺ feed for 6days. The residual Mn²⁺ level quickly reached a steady state level ofapproximately 71 μg/l, a reduction of 93% at a residence time of 21 h.

Effect of Mn²⁺ concentration on manganese removal. After operation ofthe CRFB at 1 mg/l Mn²⁺ for 6 days the manganese input was reduced to0.5 mg/l. The residual Mn²⁺ fell from 71 μg/l to 25 μg/l and withimprovement in removal performance subsequently reduced to zero after 14days (FIG. 14). Improvement in removal performance over that immediatelyafter immobilization was demonstrated by a stepwise increase in inputMn²⁺ concentrations up to 2 mg/l. The residual Mn²⁺ level remained atzero (FIG. 15).

Manganese concentration experiments were conducted over a five monthperiod. The removal performance fell away from the initial 100% toaround 90-93% for concentrations between 1 and 8.5 mg/l Mn²⁺respectively (Table 13). Over the range of 0.25 to 8.5 mg/l Mn²⁺ therewas a linear relationship between manganese concentration and themanganese removal rate (FIG. 16).

The results presented in Table 14 show that the major part of theresidual manganese was Mn²⁺ with only low levels of adsorbed Mn²⁺ andoxidised manganese in the effluent. This indicates that the majority ofthe adsorbed and oxidized Mn²⁺ remained on the immobilized cells in thecolumn.

Effect of pH on Mn²⁺ removal. A series of experiments were conducted tostudy the effect of pH conditions for manganese removal. Underuncontrolled pH conditions with a pH 7 feed, the effluent pH wasmaintained by metabolic activity around pH 7.8. Controlling pH at 7(FIGS. 17 & 18) from day 4 to day 8 resulted in cessation of Mn²⁺removal, and there was evidence of some desorption of Mn²⁺ from thecolumn as indicated by effluent Mn levels slightly above the feedconcentration. After uncontrolled pH conditions were resumed, theeffluent pH readjusted metabolically to approximately 7.6 and Mn²⁺removal returned to slightly above the initial level where the pH was7.8. The reducing conditions during control at pH 7 were also reflectedin lower Eh levels.

It appeared that manganese removal performance was favoured by alkalineconditions normally adjusted by the metabolic activity of the cells toaround pH 7.8. Control of pH at pH 8 resulted in improved Mn²⁺ and totalMn removal (FIGS. 19 & 20, Tables 15 & 16). At pH 8 the majority of theeffluent manganese was oxidised, probably as a result of the chemicaloxidation of the resifual Mn²⁺ by NaOH dosing for pH control. Control atpH7.8 reduced the chemical oxidation while only marginally affecting theMn removal rate. The reason for the drop in Eh during pH control at pH 8is uncertain at this stage.

Effect of cell age on manganese removal. The results presented in FIGS.21-24 and Table 17 show that the CRFB operated consistently over aperiod of 22 weeks. There was an initial drop in removal rate between 1and 3 months after which the removal rate remained between 92% and 93%.The pH was maintained merabolically by the cells at around pH 7.8 butbegan to drop away slightly after 20 weeks indicating a sligh loss ofmetabolic activity.

DISCUSSION

The results clearly demonstrate the successful immobilization of P.manganicum on magnetite particles and their use in a continuous recyclefluidized bioreactor for the removal of manganese from water. The CRFBoperated for 22 weeks with removal rates of greater than 90% and up to100% for Mn²⁺ concentrations up to 8.5 mg/l when operated at a residencetime of 21 h. The majority of the manganese in the effluent was residualMn²⁺ with only low levels of oxidised and adsorbed manganese. As washypothesised the bulk of the oxidised manganese remained attached to theimmobilized cells in the column. The bioreactor approached maximumremoval efficiency within a week compared with approximately 15 weeksfor sand filters relying on colonization by natural populations ofmanganese oxidizing bacteria (18). The CRFB required minimalmaintenance, did not clog or bing and therefore did not require backwashing which is a disadvantage with sand filters.

The pH conditions were critical for manganese adsorption, oxidation andremoval. Optimal conditions were found to be around pH 7.8 whichinterestingly is the pH naturally produced by P. manganicum throughmerabolic activity when grown with a feed pH of 7. Controlling the pH at7 resulted in a complete cessation of manganese oxidation. The reasonfor this dramatic impact is unclear at this stage but probably isconcerned with the machanism of manganese oxidation rather thanadsorption and desorption which is not affected by a change in pH form 8to 7 with P. manganicum (FIG. 6). Various mechanisms for manganeseoxidation have been proposed including specific enzymes or simply thecreation of local high pH environment around the cells. It is unlikelythat an enzyme would exhibit such a sharp decrease in activity. It ismore likely that controlling the pH at 7 neutralyses the alkalineenvironment around the cells thus eliminating the essential conditionsfor manganese oxidation. Further work will be required to elucidate thismatter.

The experimental results clearly show that surface components of P.manganicum are significant reservoirs of Mn²⁺. The most likely surfacecomponents are MnO₂ and extracellular polysaccharides and proteins.Viable cells of P. manganicum adsorbed more than twice the amount ofMn²⁺ compared with autoclaved cells which essentially behaved asparticulate abiological MnO₂ indicated that the extracellular componentsare heat labile. Data show that at pH8 approximately 45% of the Mn²⁺ wasadsorbed to the surface MnO₂ and 55% to the extracellular components.

The results have also shown that the extracellular components of P.manganicum stabilise the adsorption of Mn²⁺ to the cells at low pH.Whereas desorption of Mn²⁺ from abiological MnO₂ or autoclaved cells ofP. manganicum was complete at pH4, very little Mn²⁺ was desorbed fromthe viable cells at that pH.

The inhibition experiments failed to elucidate the chemical nature ofthe extracellular components. However, their adsorptive/desorptivebehaviour for Mn²⁺ can be explained in terms of the expected effect ofpH on polysaccharides which have been shown in Pedomicrobium sp. to beacidic polysaccharides [23]. Above their isoelectric point, acidicpolysaccharides are polyanionic [27] and would be expected to stronglyadsorb the oppositely charged Mn²⁺ ions. As the pH is lowered the netnegative charge on the surface of the polysaccharides would decrease andthe Mn²⁺ would be expected to desorb. Comparison of the pH desorptionprofiles for viable cells and abiological MnO₂ suggests that chargereversal occurs aroun pH4. This finding is upported by experimentalresults which show that binding of negatively charged colloidal MnO₂ tothe acidic polysaccharides of P. manganicum is enhanced by lowering thepH to 4 [50] In contrast the surface complexes formed by adsorption ofMn²⁺ ions are more easily displaced by protons.

Ghiorse and Hirsch [23] reported that bactericidal treatments ofconcentrated cell suspensions of Pedomicrobium sp. with 1 mM HgCl₂ and0.05% glutaraldehyde failed to completely inhibit manganese oxidedeposition, and that this was in conflict with the results of growthexperiments in which manganese oxidation was completely inhibited by thesame treatments. However, it is clear from our results that the abilityto adsorb Mn²⁺ is also not inhibited by the same bactericidaltreatments, not by 10 mM NaN₃ or protease enzyme. From these results itmay be concluded that although manganese oxidation is dependent onproteins, adsorption of Mn²⁺ is not. From the work of Ghiorse and Hirsch[23] it appears likely that the first step in manganese oxidation byPedomicrobium sp. is the adsorption of Mn²⁺ to acidic polysaccharides.The concept of a two stage process for microbial manganese oxidation hasbeen well established in pure culture and environmental studies[7,11,30,54] and that oxidation is the rate limiting step. The lack ofany effect on the Mn²⁺ adsorptive process of P. manganicum by proteininhibitors such as protease, HgCl₂ and glutaraldehyde, and by metabolicinhibitors such as NaN₃ suggests that the mechanism of adsorption isionic and this is supported by the pH dependence on adsorptive capacity.

The necessity when studying-manganese transformation to take intoaccount its specification and adsorption to particulate matter isessential. Various cation exchange methods have been devised to desorband estimate adsorbed Mn²⁺. It is clear from our results that not allmethods are suitable for desorption of Mn²⁺ from P. manganicum andpossibly other microorganisms probable because of the stabilisinginfluence of the extracellular acidic polysaccharides. The MgSO₄ (pH7)method performed poorly with P. manganicum desorbing 0% and 1.5% for 10mM MgSO₄ and 20 mM MgSO₄ respectively. Lowering the pH to 4 increasedthe desorption to only 2.2% and 3.4% respectively. The CuSO₄ (pH4.2)method of Bromfield and David [7] developed to desorb Mn²⁺ fromArthrobacter sp. performed very will also with P. manganicum cells andreproducibly desorbed 96% of the adsorbed Mn²⁺. The modified method [8]using 10 mM CuSO₄ in 1M ammonium acetate at pH7 was slightly lesseffective than 10 mM CuSO₄ at pH4.2. For maximum desorption from P.manganicum it was found necessary to lower the pH of the cell suspensionto pH4 before adding the CuSO₄ reagent. Increasing the CuSO₄ to 20 mMdid not increase desorption. The reason for the better performance ofCuSO₄ over MgSO₄ for ionic exchange of Mn²⁺ from the MnO₂ andextracellular polysaccharides is not clear but it is most likely due tothe properties of the acidic polysaccharides (for examply, the Cucomplexes of the carboxyl groups may be stronger than the correspondingcomplexes with Mg and Mn). An additional advantage of using CuSO₄ inmicrobial systems is the toxicity of Cu²⁺ for the manganese oxidationstep [7] which also allows the use of the reagent to stop the reactionwhen the sample is taken. Kepkay et al. [30] concluded that the CuSO₄desorption method [7] used to distinguish among adsorbed, ion-exchanged,and oxidised manganese was "approximate at best". However, Kepkay et al.[30] also concluded that there is currently no better method fordetermining the oxidation state of particulate manganese. Our resultsconfirm this conclusion for P. manganicum but have also shown that themethod is efficient and reproducible provided the sample is firstadjusted to pH4.

It is clear from this invention that the extracellular polysaccharidesof P. manganicum are an important reservoir of Mn²⁺ in the CRFB,possible accounting for more than half the Mn²⁺ on the surface ofmanganese-oxidising microbial cells in conditions where manganeseoxidation is incomplete and residual Mn²⁺ remains.

The results presented have shown that the extracellular acidicpolysaccharides of P. manganicum are also able to bind pre-formedcolloidal manganese oxide. The mechanism of MnO₂ binding to theextracellular polysaccharides is speculative at this stage but mustinvolve the surface formation of specific manganese complexes withcarbohydrate groups of the extracellular polysaccharide. The phenomenonof metal binding by polyelectrolytes has long been recognised and manyworkers have shown the complexing of divalent metal ions with polyanionsespecially those with carboxyl groups. Thus it is believed that theextracellular polysaccharide attached itself to the surface of colloidalmetal oxide particles.

The enhancement of binding as the pH was decreased suggested thatsurface charges and ionic attraction may also be involved. Publisheddate indicates that pH influences the surface charge of both δ-MnO₂(28,36,39) and extracellular acidic polysaccharides, which have a likelypKa of between 4 and 5 (21,45).

The surface sites of colloidal hydrous δ-MnO₂ have amphoteric properties(36). The isolectric point for colloidal hydrous δ-MnO₂ ranges from 2.8(36) down to 1.5 (28). Within the pH range of 4 to 7 examined in thisstudy colloidal δ-MnO₂ is negatively charged.

The extracellular polymers to which the colloidal MnO₂ was bound werestained by ruthenium red which indicated that they were polyanions innature (27). The most likely composition of these anionic polymers isacidic polysaccharides (23). It would be expected that the number ofionised carboxyl groups ton the acidic polysaccharides would bedecreased by protonation as the pH was lowered.

Consideration of the fact that the δ-MnO₂ was negatively charged underthe experimental conditions suggests that the marked increase in MnO₂binding between pH5 and 4 was due then to a decrease in the net negativecharge of the acidic polysaccharide.

The ability of P. manganicum to bind preformed MnO₂ may be used in theCRFB to remove particulate manganese from water which may pass throughsand filters.

From the foregoing it will be appreciated that the process of theinvention may also be applied to a commercial plant using a single CFRBor a multiplicity of CFRBs as shown in FIGS. 10A and 10B. The sequentialarrange of bioreactors shown in FIG. 10B may be utilised for increasedefficiency in obtaining very low levels of manganese concentration inthe purified effluent.

It will also be appreciated that operating parameters such as liquidflow rates will vary over a wide range having regard to a laboratoryscale and also having regard to a commercial scale.

We also confirm that deposit of Pedomicrobium manganicum strain UQM 3067was deposited at the Australian Government Analytical Laboratories,Suakin Street, Pymble, New South Wales, Australia on 26 Oct., 1992 andhas been allocated Accession No. 92/51402.

PUBLICATIONS

Three publications have been prepared from the results of researchundertaken in this project:

Sly, L. I., Arunpairojana, V. and Dixon, D. R. (1990) the binding ofcolloidal MnO₂ by extracellular polysaccharides of Pedomicrobiummanganicum Appl. Env. Microbiol. 56: 2791-2794.

Sly, L. I., Arunpairojana, V. and Dixon, D. R. The adsorption anddesorption of Mn²⁺ by surface components of Pedomicrobium manganicum(submitted).

Sly, L. I., Arunpairojana, V. and Dixon, D. R. The adsorption andoxidation of manganese by immobilized cells of Pedomicrobium manganicum(In preparation).

RELATED PUBLICATIONS

During the course of this project a number of papers related to thetopic were published; reports prepared, and papers delivered atconferences. These are listed for information:

Sly, L. I. (1986). Investigation into biological manganese oxidation anddepostion in the Gold Coast water distribution system. UNIQUEST Report,University of Queensland. 91 pp.

Sly, L. I. (1987). Investigation into biological manganese oxidation anddeposition in the Gold Coast water distribution system. UNIQUEST Report,University of Queensland. 57 pp.

Sly, L. I. and Arunpairojana, V. (1987). Isolation ofmanganese-oxidizing Pedomicrobium cultures from water bymicromanipulation. J. Microbiological Methods 6, 177-182.

Sly, L. I., Arunpairojana, V. and Hodgkinson, M. C. (1988).Pedomicrobium manganicum from drinking-water distribution systems withmanganese-related "dirty water" problems. Systematic and AppliedMicrobiology 11, 75-84.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1988). Effect ofwater velocity on the early development of manganese depositing biofilmin a drinking-water distribution systems. FEMS Microbiology Ecology 53,175-186.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1988). Theimportance of high aesthetic quality potable water in tourist andrecreational area. Conference on water quality and management forrecreation and tourism, Brisbane, July 1988, pp. 157-161. AustralianWater and Wastewater Association/International Association on WaterPollution Research and Control.

Sly, L. I. (1988). Abstract. Microbiological aspects of reticulatedwater. Annual Scientific Meeting. Australian Society for Microbiology,Canberra, May 1988. Australian Microbiologist 9 (2), Melbourne, Vic. p.170.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1988) Abstract.Manganese-oxidizing pedomicrobia from drinking-water distributionsystems with manganese-related "dirty-water" problems. Annual ScientificMeeting, Australian Society for Microbiology, Canberra, May 1988.Australian Microbiologist 9 (2), Melbourne, Vic. p. 245.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1989), Theimportance of high aesthetic quality potable water in tourist andrecreational areas. Water Sci. Tech. 21: 183-187.

Waite, T. D., Sly, L. I., Khoe, G. Dixon, D. R., Chiswell, B. andBarley, G. E. (1989) Manganese and iron related problems in watersupplies--observations and research needs. Proceedings Australian Waterand Wastewater Association 13th Federal Convention, Canberra, pp.437-440. The Institution of Engineers, Australia.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1989). The controlof manganese deposition and "dirty water" on the Gold Coast waterdistribution system. Proceedings Australian Water and WastewaterAssociation 13th Federal Convention, Canberra, pp. 148-151. TheInstitute of Engineers, Australia.

Dixon, D. R., Sly, L. I., Waite, T. D., Chiswell, B. and Barley, G. E.(1989). Manganese removal: A model of cooperative research. Water 15,32-34.

Sly, L. I. (1989). Deposition of manganese in the Wyong Shire waterdistribution system. UNIQUEST Report, University of Queensland. 37 p.

Sly, L. I., Chiswell, B., Hamilton, G. R., Dixon, D. R., Waite, T. D.,and Willoughby, G. A. (1989). Investigation into manganese related andother water quality problems in the Pine Rivers Shire Council watersupply. UNIQUEST Report, University of Queensland, 188 pp.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. (1990). Thedeposition of manganese in a drinking-water system. Appl. Env.Microbiol. 56: 628-639.

Sly, L. I., Hodgkinson, M. C. and Arunpairojana, V. The cause andcontrol of manganese-related "dirty water" in a drinking-waterdistribution system. (In preparation).

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                  TABLE 1                                                         ______________________________________                                        Preparations of magnetite used in the study                                                                      Surface                                                  Size      Isoelectric                                                                              area                                       Source*       (μm)   point      (m.sup.2 /g)                               ______________________________________                                        Savage River, Tasmania                                                                      104-147   5.14       0.47                                         "           147-211   5.47       0.37                                         "           211-246   5.48       0.44                                       Biggenden, Queensland                                                                       212-300   5.63       0.75                                       (Sample 1)                                                                    Biggenden, Queensland                                                                       212-300   5.82       0.73                                       (Sample 2)                                                                    ______________________________________                                         *Magnetite preparations were pretreated through 8 alternating magnetic        field (8 AMF) cycles according to the method of Mac Rae and Evans (1983).

                  TABLE 2                                                         ______________________________________                                        Effect of suspending solution on the desorption of P.                         manganicum UQM3067 adsorbed onto 1% magnetite (211-246 μm)                 after 30 seconds mixing                                                       No. cells remaining absorbed*                                                 (× 10.sup.6)                                                            Suspending solution                                                           (Mac Rae & Evans, 1983, 1984)                                                                    PC growth medium                                           ______________________________________                                        8.95               9.40                                                       9.95               10.60                                                      9.75               10.35                                                      3.50               10.65                                                      10.04              7.60                                                       9.15               9.20                                                                          9.80                                                       Mean 9.45          Mean 9.66                                                  SD ± 0.70       SD ± 1.07                                               ______________________________________                                         *The number of cells originally adsorbed was 11.4 × 10.sup.6 -          SD, Standard deviation                                                        Mean values not significantly different at P < 0.05                      

                  TABLE 3                                                         ______________________________________                                        Effect of suspending solution on the desorption of P.                         manganicum UQM3067 adsorbed onto 1% magnetite (211-246 μm)                 after 10 minutes                                                              No. cells remaining adsorbed*                                                 (× 10.sup.6)                                                            Suspending solution                                                           (Mac Rae & Evans, 1983, 1984)                                                                    PC growth medium                                           ______________________________________                                        8.4                9.6                                                        9.9                9.7                                                        7.2                8.4                                                        8.1                9.9                                                        9.3                9.95                                                       8.2                7.0                                                                           5.7                                                        Mean 8.52          Mean 8.61                                                  SD ± 0.95       SD ± 1.67                                               ______________________________________                                         *The number of cells originally adsorbed was 11.4 × 10.sup.6 -          SD, Standard deviation                                                        Mean values not significantly different at P < 0.05                      

                  TABLE 4                                                         ______________________________________                                        Effect of time on the desorption of P. manganicum UQM3067                     adsorbed onto 1% magnetite (211-246 μm) in suspending fluid                No. cells remaining adsorbed*                                                 (× 10.sup.6)                                                            After 30 seconds                                                                             After 10 minutes                                               ______________________________________                                        8.95           8.4                                                            9.95           9.9                                                            9.75           7.2                                                            8.50           8.1                                                            10.40          9.3                                                            9.15           8.2                                                            Mean 9.45      Mean 8.52                                                      SD ± 0.70   SD ± 0.95                                                   ______________________________________                                         *The number of cells originally adsorbed was 11.4 × 10.sup.6 -          SD, Standard deviation                                                        Mean values not significantly different at P < 0.05                      

                  TABLE 5                                                         ______________________________________                                        Effect of time on the desorption of P. manganicum UQM3067                     adsorbed onto 1% magnetite (211-246 μm) in PC growth medium                No. cells remaining adsorbed*                                                 (× 10.sup.6)                                                            After 30 seconds                                                                             After 10 minutes                                               ______________________________________                                        9.4            9.6                                                            10.6           9.7                                                            10.35          8.4                                                            10.65          9.9                                                            7.6            9.95                                                           9.2            7.0                                                            9.8            5.7                                                            Mean 9.66      Mean 8.61                                                      SD ± 1.07   SD ± 1.67                                                   ______________________________________                                         *The number of cells originally adsorbed was 11.4 × 10.sup.6 -          SD, Standard deviation                                                        Mean values not significantly different at P < 0.05                      

                  TABLE 6                                                         ______________________________________                                        Adsorption of P. manganicum UQM3067 by repetitive treatments                  with fresh 1% magnetite (211-246 μm)                                               No. cells No. cells         % of original                             Treatment                                                                             unadsorbed                                                                              adsorbed  % adsorbed                                                                            adsorbed                                  ______________________________________                                        1       1.77 × 10.sup.8                                                                   2.96 × 10.sup.8                                                                   62.6    62.6                                      2       7.4 × 10.sup.7                                                                    1.03 × 10.sup.8                                                                   58.2    84.4                                      3       4.5 × 10.sup.7                                                                     2.9 × 10.sup.7                                                                   39.2    90.5                                      4       3.0 × 10.sup.7                                                                     1.5 × 10.sup.7                                                                   33.3    93.7                                      ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Adsorption of Mn.sup.2+ to treated surface components of P.                   manganicum and abiological MnO.sub.2.                                                                      Mn.sup.2+ adsorbed                               Surface         Treatment    μg                                            ______________________________________                                        Pedomicrobium manganicum                                                                      Viable (aerobic)                                                                           76.3                                               "             Viable       78.7                                                             (N.sub.2 atmosphere)                                            "             1 mM HgCl.sub.2                                                                            76.8                                               "             10 mM NaN.sub.3                                                                            76.8                                               "             Protease     72.3                                                             (0.3 units/ml)                                                  "             0.05%        72.4                                                             glutaraldehyde                                                  "             2M NaOH      68.9                                               "             Autoclaved   32.7                                                             (121° C./15 min)                                         "             Steamed      29.0                                                             (100° C./10 min)                                       Abiological MnO.sub.2        30.9                                             ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of pH on Mn.sup.2+ binding capacity of particulate MnO.sub.2.          Particulate                                                                             ng Mn.sup.2+ bound per μg MnO.sub.2                              MnO.sub.2 pH8     pH7     pH6   pH5   pH4   pH3                               ______________________________________                                        P. manganicum                                                                           154.4   158.6   155.4 143.8 126.2 94.5                              (viable)                                                                      P. manganicum                                                                           70.4    50.2    27.8  9.7   0     0                                 (autoclaved)                                                                  Abiological                                                                             66.4    66.4    60.8  19.0  0     0                                 MnO.sub.2                                                                     ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Determination of Mn.sup.2+ adsorbed to P. manganicum by                       various desorption treatments.                                                                    Mn.sup.2+ desorbed (%).sup.1                              Desorption          Sample pH                                                 treatment           7        4      2                                         ______________________________________                                        pH only             0        35.1   90.5                                      10 mM MgSO.sub.4, pH7.sup.2                                                                       0        2.2    83.0                                      20 mM MgSO.sub.4, pH7.sup.3                                                                       1.5      3.4    76.8                                      10 mM CuSO.sub.4, pH4.2.sup.4                                                                     77.2     95.7   72.6                                      10 mM CuSO.sub.4    82.8     85.7   70.8                                      (in 1M ammonium acetate, pH7).sup.5                                           ______________________________________                                         .sup.1 Defined as the difference between CuSO.sub.4 -- or MgSO.sub.4--        treated cells and watertreated cells.                                         .sup.2,3,4 Treatments of Bromfield and David (7).                             .sup.5 Treatment of Bromfield and David (8).                             

                                      TABLE 10                                    __________________________________________________________________________    Effect of pH on the binding of colloidal MnO.sub.2 by cells of P.             manganicum.                                                                   Colloidal MnO.sub.2 removed*                                                  After 2 min.     After *150 min.                                                                             After 24 h.                                           With           With          With                                        Control.sup.+                                                                      cells     Control.sup.+                                                                      cells    Control.sup.+                                                                      cells                                     pH                                                                              (μg)                                                                            (μg)                                                                           % bound                                                                             (μg)                                                                            (μg)                                                                           % bound                                                                            (μg)                                                                            (μg)                                                                           % bound                               __________________________________________________________________________    4 0    204.8                                                                             54.0  10.0 342.2                                                                             88.0 36.4 243.1                                                                             54.8                                  5 0    96.6                                                                              25.6  12.2 125.7                                                                             30.1 48.5 182.3                                                                             35.4                                  6 0    33.9                                                                              9.0   16.7 63.3                                                                              12.3 42.8 80.6                                                                              10.0                                  7 0    41.0                                                                              10.9  6.0  64.7                                                                              15.5 53.8 88.5                                                                              9.2                                   __________________________________________________________________________     *Defined as MnO.sub.2 retained by filtration through a 0.45 μm membran     filter                                                                        .sup.+ Colloidal MnO.sub.2 solution without cells added                        After correction for control values                                     

                  TABLE 11                                                        ______________________________________                                        Effect to magnetite concentration on adsorption of P.                         maganicum cells                                                                      Magnetite concentration (%)                                                   1       15        30        45                                         ______________________________________                                        Inoculum 1.25 × 10.sup.10                                                                  1.25 × 10.sup.10                                                                  1.25 × 10.sup.10                                                                1.25 × 10.sup.10                   (No. of cells)                                                                No. cells                                                                              4.55 × 10.sup.9                                                                   6.04 × 10.sup.9                                                                   7.07 × 10.sup.9                                                                 8.86 × 10.sup.9                    adsorbed                                                                      % cells  36.4      48.3      56.6    70.9                                     adsorbed                                                                      cells    4.55 × 10.sup.9                                                                   4.02 × 10.sup.8                                                                   2.36 × 10.sup.8                                                                 1.97 × 10.sup.8                    adsorbed/                                                                     ml magnetite                                                                  cells/   2.06 × 10.sup.9                                                                   1.82 × 10.sup.8                                                                   1.06 × 10.sup.8                                                                 0.89 × 10.sup.8                    g magnetite.sup.a                                                             cells/   27.5       2.43      1.41    1.19                                    10.sup.4 μm.sup.2b                                                         ______________________________________                                         .sup.a 1 ml magnetite = 2.215 g dry weight                                    .sup.b 1 g magnetite has a surface area of 0.75 m.sup.2 -                

                  TABLE 12                                                        ______________________________________                                        Immobilization of P. manganicum cells on magnetite in a                       jar test model compared with the CRFB                                                        Jar test model                                                                           CFRB                                                ______________________________________                                        Magnetite concentration (%)                                                                    36           36                                              Inoculum (No. cells)                                                                           4.44 × 10.sup.10                                                                     2.17 × 10.sup.11                          No. cells adsorbed                                                                             2.85 × 10.sup.10                                                                     1.58 × 10.sup.11                          % cells adsorbed 64.2         73.0                                            Cells adsorbed/ml magnetite                                                                     5.7 × 10.sup.8                                                                      1.32 × 10.sup.8                           Cells/g magnetite.sup.a                                                                        2.57 × 10.sup.8                                                                      0.60 × 10.sup.8                           Cells/10.sup.4 μm.sup.2b                                                                     3.43         0.8                                            ______________________________________                                         .sup.a 1 ml magnetite = 2.215 g dry weight                                    .sup.b 1 g magnetite has a surface area of 0.75 m.sup.2 -                

                  TABLE 13                                                        ______________________________________                                        Removal of Mn.sup.2+ by the CRFB operated with a residence time               of 21 h                                                                                                             Removal                                 Age     Input MN.sup.2+                                                                          Residual Mn.sup.2+                                                                        %      rate                                    (months)                                                                              (mg/l)     (μg/l)   Removal                                                                              (μg/h)                               ______________________________________                                        <1      0.2553     0           100    39.8                                    <1      0.4820     0           100    76.6                                    <1      0.5142     0           100    80.2                                    <1      0.9590     0           100    153.4                                   <1      1.9070     0           100    305.1                                   3       0.2545     65.4        74.3   28.6                                    3       0.5398     69.0        87.2   71.6                                    3       1.0561     101.9       90.4   144.1                                   3       1.9620     137.8       93.0   280.9                                   3       4.7510     408.8       91.4   686.1                                   3       8.4760     581.0       93.1   1247.4                                  4       2.0250     181.0       91.1   293.2                                   5       1.9730     182.0       90.8   283.0                                   ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Conversion of Mn.sup.2+ by the CRFB operated with a residence                 time of 21 h after 3 months                                                   Input Mn.sup.2+                                                                          Effluent Mn (μg/l)                                              (mg/l)     Total     Soluble  Adsorbed MnOx                                   ______________________________________                                        0.2545     94.6      59.1     5        31                                     0.5398     116.3     64.5     7        41                                     1.0561     148.2     86.7     9        53                                     1.9620     195.3     135.3    8        52                                     4.7510     403.1     335.3    10       57                                     8.4760     534.6     471.0    11       52                                     ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Effect of pH control on Mn.sup.2+ oxidation and removal                                                             Removal                                 Controlled                                                                            Input Mn.sup.2+                                                                          Residual Mn.sup.2+                                                                        %      Rate                                    pH      (μg/l)  (μg/l)   Removal                                                                              (μg/h)                               ______________________________________                                        7.0     1986       1937        2.5    7.8                                     7.5     2001       372         81.4   255.7                                   7.8     2015       205         90.0   284.2                                   8.0     2013        48         97.6   306.5                                   ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Effect of pH control on Mn.sup.2+ conversion                                  Controlled                                                                            Input Mn.sup.2+                                                                          Effluent Mn (μg/l)                                      pH      (μg/l)  Total   Soluble                                                                              Adsorbed                                                                             MnOx                                 ______________________________________                                        7.0     1986       2010    1906   27     55                                   7.5     2001       408     360    10     41                                   7.8     2015       291     203    17     71                                   8.0     2013       202      56    12     136                                  ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        Effect of age on the removal of 2 mg/l Mn.sup.2+ by the CRFB                  operated at an influent of pH 7 and a residence time of 21 h                                                        Removal                                 Age    Input Mn.sup.2+                                                                          Residual Mn.sup.2+  Rate                                    (months)                                                                             (mg/l)     (μg/l)   % Removal                                                                             (μg/h)                               ______________________________________                                        1      1.907      0           100     305.1                                   3      1.962      137.8       93      280.9                                   4      2.025      181.0       91.1    293.2                                   5      1.973      182.0       90.8    283.0                                   ______________________________________                                    

We claim:
 1. A process for removal of manganese from water whichincludes the steps of:(i) preparing a fluidised bed of particles in abioreactor capable of adsorbing a strongly adherent biofilm ofmicroorganisms capable of metabolising manganese to provide an activelypropagated biomess; and (ii) passing a stream of water through thefluidised bed where manganese is adsorbed by said biomass and is thusremoved from the stream of water to provide a purified effluent of waterexiting from the bioreactor.
 2. A process as claimed in claim 1, whereinthe particle size of the particles in the fluidised bed is of the range50 μm to 1000 μm.
 3. A process as claimed in claim 1, wherein themicroorganisms are strains of Pedomicrobium manganicum.
 4. A process asclaimed in claim 1, wherein the microorganisms are supported onmagnetite particles.
 5. A process as claimed in claim 1, wherein thebiomass is maintained at a pH of 7.8 in step (ii).
 6. A process asclaimed in claim 4, wherein the magnetite particles have a particle sizeof approximately 200-300 μm diameter.
 7. A process as claimed in claim1, wherein the water is passed through the bioreactor continuously andalso through a mixing vessel whereby fresh influent is passed into saidmixing vessel at approximately the same rate as effluent or treatedwater is passed out of the mixing vessel to a receptacle for treatedwater.
 8. A process as claimed in claim 7, wherein the mixing vessel issubject to aeration.
 9. A process as claimed in claim 7, wherein a firstrecirculation pump is used to pump water from the bioreactor to themixing vessel.
 10. A process as claimed in claim 7, wherein a secondrecirculation pump is also used to pump water from the mixing vessel tothe bioreactor.
 11. A process as claimed in claim 1, wherein amultiplicity of bioreactors in series are utilized in step (i) whereinwater is passed from one bioreactor to provide an effluent low inmanganese concentration.
 12. A process as claimed in claim 11, wherein amixing vessel is used which is in liquid communication with eachbioreactor in the series.
 13. A process as claimed in claim 1, whereinpurified effluent is passed out of the top of the bioreactor.